Human Osteoclast Derived Cathepsin

ABSTRACT

Disclosed is a human osteoclast-derived cathepsin (Cathepsin O) polypeptide and DNA(RNA) encoding such cathepsin O polypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques. The present invention also discloses antibodies, antagonists and inhibitors of such polypeptide which may be used to prevent the action of such polypeptide and therefore may be used therapeutically to treat bone diseases such as osteoporosis and cancers, such as tumor metastases.

This application is a Division of application Ser. No. 11/185,877, filedJul. 21, 2005, which is a Division of application Ser. No. 10/726,645,filed Dec. 4, 2003, which is a Division of application Ser. No.10/114,464, filed Apr. 3, 2002, which is a Division of application Ser.No. 08/553,125, filed Nov. 7, 1995, now U.S. Pat. No. 6,475,766, whichis a Division of application Ser. No. 08/208,007, filed Mar. 8, 1994,now U.S. Pat. No. 5,501,969, each of which is herein incorporated byreference in its entirety.

STATEMENT UNDER 37 C.F.R. § 1.77(b)(5)

This application refers to a “Sequence Listing” listed below, which isprovided as a text document. The document is entitled“PF107D8_SeqList.txt” (32,888 bytes, created May 24, 2007), and ishereby incorporated by reference herein in its entirety

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention is a human osteoclast-derived cathepsin (Cathepsin O). Theinvention also relates to inhibiting the action of such polypeptide andto assays for identifying inhibitors of the polypeptide.

Bone resorption involves the simultaneous removal of both the mineraland the organic constituents of the extracellular matrix. This occursmainly in an acidic phagolysosome-like extracellular compartment coveredby the ruffled border of osteoclasts. Barron, et al., J. Cell Biol.,101:2210-22, (1985). Osteoclasts are multinucleate giant cells that playkey roles in bone resorption. Attached to the bone surface, osteoclastsproduce an acidic microenvironment between osteoclasts and bone matrix.In this acidic microenvironment, bone minerals and organic componentsare solubilized. Organic components, mainly type-I collagen, are thoughtto be solubilized by protease digestion. There is evidence that cysteineproteinases may play an important role in the degradation of organiccomponents of bone. Among cysteine proteinases, cathepsins B, L, N, andS can degrade type-I collagen in the acidic condition. Etherington, D.J. Biochem. J., 127, 685-692 (1972). Cathepsin L is the most active ofthe lysosomal cysteine proteases with regard to its ability to hydrolyzeazocasein, elastin, and collagen.

Cathepsins are proteases that function in the normal physiological aswell as pathological degradation of connective tissue. Cathepsins play amajor role in intracellular protein degradation and turnover, boneremodeling, and prohormone activation. Marx, J. L., Science. 235:285-286(1987). Cathepsin B, H, L and S are ubiquitously expressed lysosomalcysteine proteinases that belong to the papain superfamily. They arefound at constitutive levels in many tissues in the human includingkidney, liver, lung and spleen. Some pathological roles of cathepsinsinclude an involvement in glomerulonephritis, arthritis, and cancermetastasis. Sloan, B. F., and Honn, K. V., Cancer Metastasis Rev.,3:249-263 (1984). Greatly elevated levels of cathepsin L and B mRNA andprotein are seen in tumor cells. Cathepsin L mRNA is also induced infibroblasts treated with tumor promoting agents and growth factors.Kane, S. E. and Gottesman, M. M. Cancer Biology, 1:127-136 (1990).

In vitro studies on bone resorption have shown that cathepsins L and Bmay be involved in the remodelling of this tissue. These lysosomalcysteine proteases digest extracellular matrix proteins such as elastin,laminin, and type I collagen under acidic conditions. Osteoclast cellsrequire this activity to degrade the organic matrix prior to boneregeneration accomplished by osteoblasts. Several natural and syntheticinhibitors of cysteine proteinases have been effective in inhibiting thedegradation of this matrix.

The isolation of cathepsins and their role in bone resorption has beenthe subject of an intensive study. OC-2 has recently been isolated frompure osteoclasts from rabbit bones. The OC-2 was found to encode apossible cysteine proteinase structurally related to cathepsins L and S.Tezuka, K., et al., J. Biol. Chem., 269:1106-1109, (1994).

An inhibitor of cysteine proteinases and collagenase, Z-Phe-Ala-CHN₂ hasbeen studied for its effect on the resorptive activity of isolatedosteoclasts and has been found to inhibit resorption pits in dentine.Delaisse, J. M. et al., Bone, 8:305-313 (1987). Also, the effect ofhuman recombinant cystatin C, a cysteine proteinase inhibitor, on boneresorption in vitro has been evaluated, and has been shown tosignificantly inhibit bone resorption which has been stimulated byparathyroid hormone. Lerner, U. H. and Grubb Anders, Journal of Bone andMineral Research, 7:433-439, (1989). Further, a cDNA clone encoding thehuman cysteine protease cathepsin L has been recombinantly manufacturedand expressed at high levels in E. coli in a T7 expression system.Recombinant human procathepsin L was successfully expressed at highlevels and purified as both procathepsin L and active processedcathepsin L forms. Information about the possible function of thepropeptide in cathepsin L folding and/or processing and about thenecessity for the light chain of the enzyme for protease activity wasobtained by expressing and purifying mutant enzymes carrying structuralalterations in these regions. Smith, S. M. and Gottesman, M. M., J. BioChem., 264:20487-20495, (1989). There has also been reported theexpression of a functional human cathepsin S in Saccharomyces cerevisiaeand the characterization of the recombinant enzyme. Bromme, D. et al.,J. Bio Chem., 268:4832-4838 (1993).

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is a osteoclast-derivedcathepsin as well as fragments, analogs and derivatives thereof. Thehuman osteoclast-derived cathepsin of the present invention is of humanorigin.

In accordance with another aspect of the present invention, there areprovided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with still another aspect of the present invention, thereis provided a procedure for producing such polypeptide by recombinanttechniques.

In accordance with yet a further aspect of the present invention, thereis provided an antibody which inhibits the action of such polypeptide.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, e.g., a small moleculeinhibitor which may be used to inhibit the action of such polypeptide,for example, in the treatment of metastatic tumors and osteoporosis.

In accordance with still another aspect of the present invention, thereis provided a procedure for developing assay systems to identifyinhibitors of the polypeptide of the present invention.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are meant only as illustrations of specificembodiments of the present invention and are not meant as limitations inany manner.

FIGS. 1A-B show the polynucleotide sequence (SEQ ID NO:1) andcorresponding deduced amino acid sequence (SEQ ID NO:2) for cathepsin O.The cathepsin O shown is the predicted precursor form of the proteinwhere approximately the first 15 amino acids represent the leadersequence and the first 115 amino acids are the prosequence. The standardthree letter abbreviation has been used for the amino acid sequence.

FIGS. 2A-C are illustrations of the amino acid homology of cathepsin Oto other human cathepsins (SEQ ID NO:8-14) and rabbit OC-2 (SEQ IDNO:7).

In accordance with one aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) which encodes for themature polypeptide having the deduced amino acid sequence of FIGS. 1A-B(SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of theclone deposited as ATCC™ Deposit No. 75671 on Feb. 9, 1994.

The ATCC™ number referred to above is directed to a biological depositwith the ATCC™ (American Type Culture Collection), 10801 UniversityBoulevard, Manassas, Va. 20110-2209. Since the strains referred to arebeing maintained under the terms of the Budapest Treaty, they will bemade available to a patent office signatory to the Budapest Treaty.

A polynucleotide encoding a polypeptide of the present invention may beobtained from a cDNA library derived from human osteoclastoma cells,placenta, kidney or lung. The polynucleotide described herein wasisolated from a cDNA library derived from human osteoclastoma cells. ThecDNA insert is 1619 base pairs (bp) in length and contains an openreading frame encoding a protein 329 amino acids in length of whichapproximately the first 15 amino acids represent the leader sequence andfirst 115 amino acids represent the prosequence. Thus, the mature formof the polypeptide of the present invention consists of 214 amino acidsafter the 115 amino acid prosequence (which includes the approximately15 amino acid leader sequence) is cleaved. The polypeptide encoded bythe polynucleotide is structurally related to human cathepsin S with 56%identical amino acids and 71% similarity over the entire coding region.It is also structurally related to rabbit OC-2 cathepsin with 94%identical amino acids and 97% similarity over the entire coding region.The polypeptide may be found in lysosomes of, or extracellularly near,osteoclasts.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIGS. 1A-B (SEQ ID NO:1) or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same, mature polypeptide as the DNA of FIGS. 1A-B (SEQID NO:1) or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIGS.1A-B (SEQ ID NO:2) or for the mature polypeptide encoded by thedeposited cDNA may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide andadditional coding sequence such as a leader or secretory sequence or aproprotein sequence; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs, andderivatives of the polypeptide having the deduced amino acid sequence ofFIGS. 1A-B (SEQ ID NO:2) or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide. The present invention alsorelates to polynucleotide probes constructed from the polynucleotidesequence of FIGS. 1A-B (SEQ ID NO:1) or a segment of the sequence ofFIGS. 1A-B (SEQ ID NO:1) amplified by the PCR method, which could beutilized to screen an osteoclast cDNA library to deduce the polypeptideof the present invention.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIGS. 1A-B (SEQ ID NO:2) or the samemature polypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIGS. 1A-B (SEQ ID NO:2) orthe polypeptide encoded by the cDNA of the deposited clone. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 1A-B (SEQ ID NO:1) or of the coding sequence of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and may in some cases be an inactive formof the protein. Once the prosequence is cleaved an active mature proteinremains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a presequence (leader sequence) and aprosequence.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term “stringent conditions” means hybridization will occur only ifthere is as least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of FIGS. 1A-B or thedeposited cDNA.

The deposits referred to herein will be maintained under the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the purposes of Patent Procedure. These deposits are provided merelyas a convenience and are not an admission that a deposit is requiredunder 35 U.S.C. § 112. The sequence of the polynucleotides contained inthe deposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with the description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

The present invention further relates to a cathepsin O polypeptide whichhas the deduced amino acid sequence of FIGS. 1A-B (SEQ ID NO:2) or whichhas the amino acid sequence encoded by the deposited cDNA, as well asfragments, analogs and derivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIGS. 1A-B (SEQ ID NO:2) or that encoded by the depositedcDNA, means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIGS. 1A-B (SEQID NO:2) or that encoded by the deposited cDNA may be (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the cathepsin O genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

The polynucleotide of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, for example,the polynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,and pseudorabies. However, any other plasmid or vector may be used aslong as it is replicable and viable in the host.

As hereinabove indicated, the appropriate DNA sequence may be insertedinto the vector by a variety of procedures. In general, the DNA sequenceis inserted into appropriate restriction endonuclease sites byprocedures known in the art. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain a gene to providea phenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium; Streptomyces; fungal cells, such as yeast; insect cellssuch as Drosophila and Sf9; animal cells such as CHO, COS or Bowesmelanoma; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen)pBs, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a,pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as it is replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacd, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, 1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), thedisclosure of which is hereby incorporated by reference.

Transcription of a DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication origin,and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC™ 37017).Such commercial vectors include, for example, PKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,U.S.A.). These pBR322 “backbone” sections are combined with anappropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell-known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 viralgenome, for example, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

Cathepsin O is recovered and purified from recombinant cell cultures bymethods used heretofore, including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxyapatite chromatography and lectin chromatography. It is preferredto have low concentrations (approximately 0.1-5 mM) of calcium ionpresent during purification (Price, et al., J. Biol. Chem., 244:917(1969)). Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be naturally purifiedproducts expressed from a high expressing cell line, or a product ofchemical synthetic procedures, or produced by recombinant techniquesfrom a prokaryotic or eukaryotic host (for example, by bacterial, yeast,higher plant, insect and mammalian cells in culture). Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated with mammalian or othereukaryotic carbohydrates or may be non-glycosylated. Polypeptides of theinvention may also include an initial methionine amino acid residue.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphism's) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 500 or 600bases; however, clones larger than 2,000 bp have a higher likelihood ofbinding to a unique chromosomal location with sufficient signalintensity for simple detection. FISH requires use of the clone fromwhich the EST was derived, and the longer the better. For example, 2,000bp is good, 4,000 is better, and more than 4,000 is probably notnecessary to get good results a reasonable percentage of the time. For areview of this technique, see Verma et al., Human Chromosomes: a Manualof Basic Techniques. Pergamon Press, N.Y. (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man (available on line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and diseases that have been mapped to the same chromosomal regionare then identified through linkage analysis (coinheritance ofphysically adjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that cDNA sequence. Ultimately, completesequencing of genes from several individuals is required to confirm thepresence of a mutation and to distinguish mutations from polymorphisms.

The present invention is directed to inhibiting cathepsin O in vivo bythe use of antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the polynucleotidesequence, which encodes for the mature polypeptide of the presentinvention, is used to design an antisense RNA oligonucleotide of from 10to 40 base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix—see Lee et al, Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al, Science, 251:1360 (1991),thereby preventing transcription and the production of cathepsin O. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of an mRNA molecule into the cathepsin O (antisense—Okano,J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).

Alternatively, the oligonucleotides described above can be delivered tocells by procedures in the art such that the anti-sense RNA or DNA maybe expressed in vivo to inhibit production of cathepsin O in the mannerdescribed above.

Antisense constructs to cathepsin O, therefore, inhibit the action ofcathepsin O and may be used for treating certain disorders, for example,osteoporosis, since bone resorption is slowed or prevented. Theseantisense constructs may also be used to treat tumor metastasis sinceelevated levels of cathepsins are found in some tumor cells, andcathepsin L mRNA and protein is increased in ras-transformedfibroblasts. Further, there is evidence that metastatic B16 melanomasall upregulate cathepsin B compared with non-metastatic tumors.

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present also includes chimeric, single chainand humanized antibodies, as well as Fab fragments, or the product of anFab expression library. Various procedures known in the art may be usedfor the production of such antibodies and fragments.

Antibodies generated against the polypeptide corresponding to a sequenceof the present invention or its in vivo receptor can be obtained bydirect injection of the polypeptide into an animal or by administeringthe polypeptide to an animal, preferably a nonhuman. The antibody soobtained will then bind the polypeptide itself. In this manner, even asequence encoding only a fragment of the polypeptide can be used togenerate antibodies binding the whole native polypeptide. Suchantibodies can then be used to isolate the polypeptide from tissueexpressing that polypeptide. For preparation of monoclonal antibodies,any technique which provides antibodies produced by continuous cell linecultures can be used. Examples include the hybridoma technique (Kohlerand Milstein, 1975, Nature, 256:495-497), the trioma technique, thehuman B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today4:72), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole, et al., 1985, in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention.

Antibodies specific to the cathepsin O may further be used to inhibitthe biological action of the polypeptide by binding to the polypeptide.In this manner, the antibodies may be used in therapy, for example, totreat cancer since cathepsin L mRNA and protein is increased inras-transformed fibroblasts and after addition of phorbol esters andgrowth factors. Also, osteoporosis may be treated with these antibodiessince bone resorption by cathepsin O is prevented.

Further, such antibodies can detect the presence or absence of cathepsinO and the level of concentration of cathepsin O and, therefore, areuseful as diagnostic markers for the diagnosis of disorders such as highturnover osteoporosis, Paget's disease, tumor osteolysis, or othermetabolic bone disorders. Such antibodies may also function as adiagnostic marker for tumor metastases.

The present invention is also directed to antagonists and inhibitors ofthe polypeptides of the present invention. The antagonists andinhibitors are those which inhibit or eliminate the function of thepolypeptide.

Thus, for example, an antagonist may bind to a polypeptide of thepresent invention and inhibit or eliminate its function. The antagonist,for example, could be an antibody against the polypeptide whicheliminates the activity of cathepsin O by binding to cathepsin O, or insome cases the antagonist may be an oligonucleotide. An example of aninhibitor is a small molecule inhibitor which inactivates thepolypeptide by binding to and occupying the catalytic site, therebymaking the catalytic site inaccessible to a substrate, such that thebiological activity of cathepsin O is prevented. Examples of smallmolecule inhibitors include but are not limited to small peptides orpeptide-like molecules.

In these ways, the antagonists and inhibitors may be used to treat bonedisease, such as osteoporosis by preventing cathepsin O from functioningto break down bone. The antagonists and inhibitors may also be used totreat metastatic tumors since cathepsins play a role in increasingmetastatic tumor growth.

The antagonists and inhibitors may be employed in a composition with apharmaceutically acceptable carrier, including but not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol andcombinations thereof. Administration of cathepsin inhibitors arepreferably systemic. Intraperitoneal injections of the cysteineproteinase inhibitor leupeptin (0.36 mg/kg body weight) and E-64 (0.18mg/kg body weight) in rats were able to decrease serum calcium andurinary excretion of hydroxyproline. Delaisse et al., BBRC, 125:441-447(1984). A direct application on areas of bone vulnerable to osteoporosissuch as the proximal neck of the femur may also be employed.

The present invention also relates to an assay for identifying theabove-mentioned small molecule inhibitors which are specific toCathepsin O and prevent it from functioning. Either natural proteinsubstrates or synthetic peptides would be used to assess proteolyticactivity of cathepsin O, and the ability of inhibitors to prevent thisactivity could be the basis for a screen to identify compounds that havetherapeutic activity in disorders of excessive bone resorption.Maciewicz, R. A. and Etheringtin, D. J., BioChem. J. 256:433-440 (1988).

A general example of such an assay for identifying inhibitors ofcathepsin O utilizes peptide-based substrates which are conjugated witha chromogenic tag. An illustrative example of such a peptide substratehas the X—(Y)_(n)-Z, wherein X represents an appropriate aminoprotecting group such as acetyl, acetate or amide, Y is any naturally ornon-naturally occurring amino acid which in combination forms asubstrate which cathepsin O recognizes and will cleave in the absence ofan inhibitor, n represents an integer which may be any number, however,which is usually at least 20, and Z represents any chromogenic orflourogenic tag, for example, para-nitroanelide or n-methyl coumarin,which upon cleavage of the Y group by the cathepsin O can be monitoredfor color production. If the potential inhibitor does not inhibitcathepsin O and the substrate (Y group) is cleaved, Z has acorresponding change in configuration, which change allows fluorescenceto be detected by a fluorimeter in the case of a flourogenic tag andcolor to be detected by a spectrophotometer in the case of a chromogenictag. When the inhibitor successfully inhibits cathepsin O from cleavingthe substrate, the Y group is not cleaved and Z does not have a changein configuration and no fluorescence or color is detectable whichindicates that the inhibitor has inhibited the action of cathepsin O.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples, certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe methods of Graham, F. and Van Der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Expression and Purification of the Osteoclast-DerivedCathepsin

The DNA sequence encoding for cathepsin O (ATCC™ #75671) is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ end of the DNA sequence to synthesize insertion fragments. The 5′oligonucleotide primer has the sequence 5′ GCTAAGGATCCTGGGGGCTCAAGGTT 3′(SEQ ID NO:3) contains a Bam H1 restriction enzyme site followed by 15nucleotides of cathepsin O coding sequence starting from the codonfollowing the methionine start codon; the 3′ sequence, 5′GCTAATCTAGATCACATCTTGGGGAA 3′ (SEQ ID NO:4) contains complementarysequences to XbaI site, and the last 12 nucleotides of cathepsin Ocoding sequence. The restriction enzyme sites correspond to therestriction enzyme sites on the bacterial expression vector pQE-9(Qiagen Inc., 9259 Eton Ave., Chatsworth, Calif. 91311). The plasmidvector encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter/operator (P/O), aribosome binding site (RBS), a 6-histidine tag (6-His) and restrictionenzyme cloning sites. The pQE-9 vector was digested with Bam HI and XbaIand the insertion fragments were then ligated into the vectormaintaining the reading frame initiated at the bacterial RBS. Theligation mixture was then used to transform the E. coli strain m15/rep4(available from Qiagen under the trademark m15/rep4). M15/rep4 containsmultiple copies of the plasmid pREP4, which expresses the lacd repressorand also confers kanamycin resistance (Kan^(r)). Transformants areidentified by their ability to grow on LB plates containing both Amp andKan. Clones containing the desired constructs were grown overnight (O/N)in liquid culture in either LB media supplemented with both Amp (100μg/ml) and Kan (25 μg/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity of 600 (O.D.⁶⁰⁰) between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacd repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 3-4 hours. Cells were then harvested by centrifugation. Thecell pellet was solubilized in the chaotropic agent 6 molarguanidine-HCL and 50 mM NaPO₄ pH 8.0. After clarification, solubilizedcathepsin O was purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag. (Hochuli, E. et al., GeneticEngineering, Principle & Methods, 12:87-98 Plenum Press, New York(1990)). Cathepsin O (95% pure) was eluted from the column in 6 molarguanidine-HCL, 150 mM NaPO₄ pH 5.0.

EXAMPLE 2 Expression Pattern of Cathepsin O in Human Tissue

[³⁵S]-labeled sense or antisense riboprobes generated from a partialcDNA clone of Cathepsin O were used as part of a Northern blot analysisto probe cryosections of osteoclastoma tissue, which demonstrated asingle mRNA species, and spleen tissue. Current Protocols in MolecularBiology, Vol. 2, Ausubel et al., editors, section 14.3. Total RNA wasisolated from osteoclastoma tissue and spleen. The RNA waselectrophoresed on a formaldehyde agarose gel, and transferred tonitrocellulose. Following pre-hybridization, the blot was hybridizedovernight with either sense or antisense [³²P]-labeled riboprobe at2×10⁶ cpm/ml at 42° C. Following stringent washes (0.2×SSC at 65° C.),the blots were exposed to x-ray film. When used in in situ hybridizationon sections of osteoclastoma tissue, specific, high level expression wasobserved in the osteoclasts; some expression was observed in mononuclearcells, but the stromal cells and osteoblasts did not express the mRNAfor Cathepsin O at detectable levels. When sections of spleen tissuewere used for in situ hybridization, no expression of Cathepsin O wasobserved. These data indicate that the mRNA for Cathepsin O is expressedat high levels in osteoclasts, and appears to be selectively expressedin these cells.

EXAMPLE 3 Analysis of Cathepsin O Using Antibodies

Antibodies were prepared against synthetic peptides from the Cathepsin Osequence, from regions sufficiently dissimilar to other members of thecathepsin family to allow specific analysis of Cathepsin O in Westernblots. The antibodies were affinity purified and used to probe Westernblots of osteoclastoma tissue. Synthetic peptides (AIDASLTSFQFYSK (SEQID NO:5) and YDESCNSDNLN (SEQ ID NO:6)) were prepared based upon thepredicted sequence of Cathepsin O (corresponding to amino acids 248-261and 265-275 in FIG. 1). The regions were chosen because of lowestidentity to other members of the cathepsin family. The peptides wereconjugated to Keyhole Limpet Hemocyanin with glutaraldehyde, mixed withadjuvant, and injected into rabbits. Immune sera was affinity purifiedusing the immobilized peptide. Drake et al., Biochemistry, 28:8154-8160(1989).

Tissue samples were homogenized in SDS-PAGE sample buffer and run on a14% SDS-PAGE. The proteins were transferred to nitrocellulose, followedby blocking in bovine serum albumin. Immunoblotting was carried out withaffinity purified anti-peptide antibodies, followed by alkalinephosphatase conjugated second antibody and visualization with achromogenic substrate. Molecular mass determination was made based uponthe mobility of pre-stained molecular weight standards (Rainbow markers,Amersham). Antibodies to two different peptides recognized a major bandof approximately 29 kDa and a minor band of approximately 27 kDa. Theimmunoreactivity could be competed by the peptides used to generate theantibodies, confirming the specificity of the signal. This indicatesthat the mRNA for Cathepsin O is actually expressed in the tissue, andproduces a protein with a size consistent with that of a fully processedCathepsin O (assuming processing similar to related cathepsins).

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1. An isolated polynucleotide encoding for Cathepsin O, saidpolynucleotide selected from the group consisting of (a) apolynucleotide encoding for the Cathepsin O polypeptide having thededuced amino acid sequence of FIG. 1 or a fragment, analog orderivative of said polypeptide; and (b) a polynucleotide encoding forthe Cathepsin O polypeptide having the amino acid sequence encoded bythe cDNA contained in ATCC™ Deposit No. 75671 or a fragment, analog orderivative of said polypeptide.
 2. The polynucleotide of claim 1 whereinthe polynucleotide is DNA.
 3. The polynucleotide of claim 2 having thecoding sequence for Cathepsin O deposited as ATCC™ Deposit No.
 75671. 4.A vector containing the DNA of claim
 2. 5. A host cell geneticallyengineered with the vector of claim
 4. 6. A process for producing apolypeptide comprising: expressing from the host cell of claim 5 thepolypeptide encoded by said DNA.
 7. A process for producing cellscapable of expressing a polypeptide comprising genetically engineeringcells with the vector of claim
 4. 8. An isolated DNA hybridizable to theDNA of claim 2 and encoding a polypeptide having Cathepsin O activity.9. A polypeptide selected from the group consisting of (i) a Cathepsin Opolypeptide having the deduced amino acid sequence of FIG. 1 andfragments, analogs and derivatives thereof and (ii) a Cathepsin Opolypeptide encoded by the cDNA of ATCC™ Deposit No. 75671 andfragments, analogs and derivatives of said polypeptide.
 10. Thepolypeptide of claim 9 wherein the polypeptide is Cathepsin O having thededuced amino acid sequence of FIG.
 1. 11. An antibody against thepolypeptide of claim
 9. 12. An antagonist/inhibitor against thepolypeptide of claim
 9. 13. A method for the treatment of a patienthaving need to inhibit Cathepsin O comprising: administering to thepatient a therapeutically effective amount of an antagonist against thepolypeptide of claim
 9. 14. A pharmaceutical composition comprising thepolypeptide of claim 9 and a pharmaceutically acceptable carrier.
 15. Amethod for the treatment of a patient having need to inhibit Cathepsin Ocomprising: administering to the patient a therapeutically effectiveamount of an antisense construct against the DNA or RNA which encodesfor Cathepsin O such that transcription and translation into Cathepsin Ois inhibited.
 16. A method for the treatment of a patient having need toinhibit Cathepsin O comprising: administering to the patient atherapeutically effective amount of the antibody of claim
 11. 17. Amethod for the treatment of a patient having need to inhibit Cathepsin Ocomprising: administering to the patient a therapeutically effectiveamount of the antagonist/inhibitor of claim
 12. 18. The method of claim16, wherein said patient has cancer.
 19. A method of detecting CathepsinO protein in a biological sample comprising: (a) contacting thebiological sample with the antibody of claim 11; and (b) detecting theCathepsin O protein in the biological sample.